Here is a video of us outlining our experiment. It can be used as an outline for anyone who wishes to continue this project once we graduate this year (May 2014). https://www.youtube.com/watch?v=96rwsh61T3s&feature=youtu.be

We've come to the end of this experiment. We ran the 13 samples we had through PCR and gel electrophoresis. Unfortunately, there were complications that prohibited us from seeing the resulting DNA within the gel. This could be due to human error during PCR, where the DNA was not replicated properly or not at all, or an improper stain balance within the gel. In the most recent trial, we used 5.5µL of Syber Safe gel in combination with the agarose. We used a heightened amount because our last gel, with the 1:1µL ratio, did not present DNA bars either. However, the first gel presented the DNA ladder, therefore proving that the if you use more Syber Safe, the DNA will not necessarily become clearer.

Beyond this particular experiment, Alexandra Van Pernis and I are in a new Biotechnology class at HPA. In this class we are working with the National Barcode of Life project to help genome every species on Earth. The project is working to classify fish right now, so that is what our Biotech class is working on. So far we have amplified the gDNA in the fish cell samples the program sent us. We have also selected the CO1 barcode gene located in the mitochondria. After we performed gel electrophoresis to ensure we had, in fact, located and isolated the gene (picture below), we sent our samples off to the lab to be sequenced. The next step in our project will be to analyze the completed sequence and publish our findings. Our names will be placed next to the sample for future generations to see. More information on the project itself can be found here: http://www.educationandbarcoding.org/index.php# . Recently, Alexandra and I were featured in West Hawaii Today in an article about our new class, http://westhawaiitoday.com/news/local-features/biotechnology-class-hpa-first-state-identify-genetic-code-global-genomics .

In our last work period (March 7th), we performed gel electrophoresis with the gel we created last class and seven of our current DNA samples. The gel rejected (the samples floated out of their wells) the first few samples when we placed them in the wells. We believe this is because the gel was kept in a refrigerator that was set at too low a temperature. Having gels too cold cause cause them to shrivel and compress. This compression leaves the wells very shallow, meaning that the samples cannot properly sit there. Out of the 8 wells, only the first three had this issue so we hoped that a few of our samples would be left unaffected after electrophoresis. After the thirty minute wait, we used our blue light transilluminator to examine the gel. Only the DNA ladder (surprisingly in one of the three affected wells) appeared. We have a few hypotheses as to why our DNA did not fluoresce under the blue light. The first idea is that we used an improper amount of Syber Safe DNA stain when casting the gel. The second is that we added the stain to the in process gel before heating it, when we should have added it after. Either of these are plausible conclusions to our failed experiment. Nevertheless, we have 6 samples remaining before we must venture out to collect more. Our next test will be conducted after spring break with these 6 samples. Hopefully it will work. Below is a picture of our gel after electrophoresis. The only bands that are evident (towards the upper left corner) are from the DNA ladder.

We have brought our samples through PCR. We are currently in the process of casting our agarose gels for gel electrophoresis. Because we are constructing these gels with the new Syber Safe DNA stain, I have included a procedure for casting below.

ONE GEL requires approximately:

30 microliters (µL) of TBE buffer

3 microliters (µL) of Syber Safe DNA stain (**approximately one µL of stain is required for every 10 µL of TBE buffer used)

0.6 grams of agarose powder (if increasing amount, keep the agarose powder at a 2% mixture with the TBE buffer)

Procedure:

1. Measure out 30 µL of TBE buffer in a graduated cylinder.

2. Measure out 0.6 grams of agarose powder on a sensitive scale.

3. Combine TBE buffer and agarose powder in a small flask. DO NOT MIX

4. Add 3 µL of Syber Safe DNA stain to the flask. DO NOT MIX

5. Plug the top of the flask with Kim wipes, to avoid overflows during the boiling process.

6. Microwave in small 10-15 second increments until the liquid in flask seems clear or before it begins to spill over.

7. Allow flask to cool until it is safe to touch.

8. Pour mixture into a gel casting tray.

9. Allow to solidify. (Could take upwards of 20 minutes)

10. Remove sample comb and side stoppers.

We have finally gotten our PCR beads in. Next work period we will be bringing our 14 samples through the first PCR process. This step will select the ACTN3 gene and amplify it thousands of times over. After this step we will be able to perform our gel electrophoresis and actually see the results of this experiment. We have also recently been featured in "West Hawaii Today" under an article about the energy lab. (Picture above) The article outlines our experiment, found here http://westhawaiitoday.com/news/local-features/energy-lab-recognized-apple-distinguished-program

We are still waiting on our PCR beads. It has been over a month since we ordered them at the beginning of January. We have emailed the company and they have responded saying the beads are on backorder. They are expected to arrive within the week, hopefully.

We have collected 14 samples are are ready to progress in our experiment. However, as we were ready to start PCR, we realized we are out of ready-to-go PCR beads. Without these, the three steps of PCR (denaturing, annealing, and extension) will not happen properly. We have ordered the beads and they are expected to arrive in the middle of this month. (January)

We are beginning our ATCN3 gene test among the athletes at HPA. As of now (January, 10th) we have approximately 6 DNA collections. We hope to have 15 by the end of this test in order to get a more widespread test base. Collecting the DNA has been harder than anticipated as not many people who were randomly selected have replied to our email. We have to go and seek them out instead. We will begin the next step in our process once we collect all 15 samples.

We finished our last round of testing for the year with the new Syber Safe gel stain. However, our dye ratio was off for the amount of agarose gel we used and the stain only clung to the DNA ladder which is the comparative substance. None of our DNA was exemplified. Below is a picture taken of our blue light transilluminator with our gel. The visible lines are the bars of DNA from the DNA ladder.

We are currently running our last test for the year. It is a PTC taste gene test. While we are hoping to start our fast twitch muscle gene with the new year, we need a DNA ladder for this new gene. In addition, running further PTC taste gene tests will ensure that we are familiar with the new dye. Below is the gel being run through gel electrophoresis.

We are going through the first round of our new experiment for the fast twitch muscle gene. So far we have 6 DNA samples that have all been brought through the first step in analyzation. Now that we have our loading dye in (enough for the rest of the year), we can bring the samples through the remainder of the testing and record our results. Hopefully, we will find an interesting correlation between the type of sport and the presence of the fast twitch muscle gene.

We did an inventory the other day and all we are waiting on now is our loading dye. Once we get that in, we can begin working with similar previous experiments to find out which enzymes will be most useful in isolating the fast twitch muscle gene. At the end of that process, we can begin the testing itself, beginning with collecting DNA from our subjects.

Our Syber Safe DNA stain finally came in. With this, we can use our blue light transilluminator to look at the bands of DNA in our electrophoresis gels. 400 microliters may not seem like much but we only need one microliter for every gel, so that small amount will go a long way. Now, all we are waiting for is our agarose powder. Once we get that in, we can begin collecting DNA from our new subjects, all from different types of sports, to test for the fast twitch muscle gene.

We are trying to figure out what correlations we should test between sports and the fast twitch muscle gene. As of now we have three categories to test: Endurance, Contact, and Explosive. Endurance sports include cross country and long distance swimming. Contact sports are football, wrestling, and water polo. Lastly, explosive sports are basketball, baseball, volleyball, and non-distance track. We will be testing people who perform 0, 1,2, or 3 of these sports for the fast twitch muscle gene. We will be able to publish our findings and show a correlation, if any, between specific sports and this gene.

Some of our new materials came in for our experiment. These include our buffer and enzyme for the fast twitch muscle gene. We are still missing our agarose powder and have recently realized we are out of loading dye. Hopefully both of these will come in soon so we can begin our experiments.

We've been working on our new experiment plan for fast twitch muscle gene. Since there is no set experimental design, we have to create our own. Through research and previous experiments we have come up with a rough outline. The materials we need are the Syber Safe stain, Agarose powder, 1xNEB3 Buffer, and 1 microlitre Dde I. We are trying to find nucleotide primers which will cut the DNA at the forward 5' CTG TTG CCT GTG GTA AGT GGG 3' mark and the reverse 5' TGG TCA CAG TAT GCA GGA GGG 3' mark. We know the fragment length R577- 205bp, 86bp/ 577X- 108bp, 976bp, 86bp/ R577X- a08bp, 97bp, 86bp. Besides that, we have found the PCR sequences for the experiment. The initial denaturing of the DNA will occur at 95ºC for 5 minutes. This is followed by 35 cycles, each cycle at 95ºC for 30 seconds, 60ºC for 30 seconds, and 72ºC for 30 seconds. The final extension will be at 72ºC for 10 minutes with one hour incubation at 37ºC. Hopefully we can get this started soon!

We just ordered our new stain to go along with our blue light transilluminator. This stain is non-toxic and highly efficient. We only need one microliter per electrophoresis gel. Hopefully it, along with our new materials, will come in soon so we can begin our testing with the fast twitch muscle gene.

We got in our blue light transilluminator! It is designed to replace the highly toxic ethidium bromide we are currently using. Instead, it uses orange lenses along with a new dye to show the DNA strands in the electrophoresis gel. We can't wait to utilize it in our next experiment.